Poly(beta malic acid) with pendant leu-leu-leu tripeptide for effective cytoplasmic drug delivery

ABSTRACT

The invention relates to the use of Polycefin-LLL nanoconjugate as a means of cytoplasmic delivery of drugs. In one embodiment, the present invention provides a drug delivery molecule, comprising a polymerized carboxylic acid molecular scaffold covalently linked to L-leucylleucylleucine. In another embodiment, the Polycefin-LLL includes drug antisense morpholino oligos, targeting antibodies, and a pH-sensitive endosome escape unit. In addition, the drug could be siRNA, microRNA, and aptamer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Phase of International ApplicationPCT/US09/40252, filed Apr. 10, 2009, which designated the U.S. and thatInternational Application was published under PCT Article 21(2) inEnglish. This application also includes a claim of priority under 35U.S.C. §119(e) to U.S. provisional, patent application No. 61/044,191filed Apr. 11, 2008.

GOVERNMENT RIGHTS

This invention was made with government support under Grant Nos.R01CA123495, R01CA136841 and U01CA151815 awarded by the National CancerInstitute. The government may have certain rights in the invention.

BACKGROUND

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thefollowing description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Treatments using nucleic acid-based drugs such as DNA, antisenseoligonucleonucleotides, small interfering RNA (siRNA), micro-RNA and/oraptamers are attractive but sometimes challenging. For example, nucleicacid-based drugs that specifically target oncogenes have the potentialto block the unique initiation and propagation pathways of cancer cells,but their clinical application can be hindered due to the lack of safeand efficient drug delivery systems. Besides being non-toxic andnon-immunogenic, an ideal drug delivery system for nucleic acid-basedtherapeutics must be able to assist the escape of therapeutics from theendosome so that the nucleic acid-based drug can be released into thecytoplasm where it can function.

Previously, it was believed that direct cytoplasmic delivery may beachievable using cell penetrating peptides (CPPs) which translocateacross the cell membrane, thus bypassing the barrier of endosome anddirectly delivering their cargo into the cytoplasm (Elliott, G.; Cell1997, 88, (2), 223-33). However, additional studies revealed that theinternalization of most CPPs actually involve endocytosis (Trehin, R.;Eur J Pharm Biopharm 2004, 58, (2), 209-23). Moreover, most biomoleculesare internalized by cells through endocytosis and are subsequentlychanneled into endosomal-lysosomal compartments, resulting in total lossof activity in lysosome if they do not possess the machinery to escapefrom the endosome. Thus, the endosome is a major barrier for cytoplasmicdelivery of nucleic acid-based therapeutics and drug delivery systemsmust be designed to destabilize the endosome membrane to assist theescape of the therapeutics.

Thus, there is a need in the art for an effective cytoplasmic drugdelivery system that can destabilize the endosome membrane.

SUMMARY OF THE INVENTION

Various embodiments include a drug delivery system, comprising apolymerized carboxylic acid molecular scaffold covalently linked to oneor more biologically active molecular modules, where one of the one ormore biologically active molecular modules comprises an endosome escapeunit. In another embodiment, the polymerized carboxylic acid molecularscaffold is covalently linked to one or more biologically activemolecular modules by a cleavable disulfide bond. In another embodiment,the endosome escape unit comprises L-leucylleucylleucine (LLL). Inanother embodiment, the endosome escape unit is pH sensitive. In anotherembodiment, one of the one or more biologically active molecular modulescomprises an antisense Morpholino oligonucleotide. In anotherembodiment, one of the one or more biologically active molecular modulescomprises an siRNA, a microRNA, and/or an aptamer. In anotherembodiment, one of the one or more biologically active molecular modulescomprises at least one targeting module for promoting cellular uptake.

Other embodiments include a method of delivering a nucleic acid-basedtherapeutic to the cytoplasm of a cell, comprising providing acomposition comprising a nucleic acid-based therapeutic covalentlylinked by cleavable disulfide bonds to a drug delivery system designedto destablize an endosome membrane, and administering an amount of thecomposition effective to achieve a certain quantity of the nucleicacid-based therapeutic to the cell. In another embodiment, the cell is aglioma cell. In another embodiment, the drug delivery system comprisespoly(β-L-malic acid) (PMLA) containing about 40% of a pendantcarboxylate conjugated by an amide bond. In another embodiment, thependent carboxylate comprises trileucine.

Other embodiments include a method of synthesizing a drug deliverymolecule, comprising providing a quantity of a Leu-Leu-Leu tripeptide,providing a quantity of a polymalic acid, and conjugating theLeu-Leu-Leu tripeptide with the polymalic acid. In another embodiment,the polymalic acid comprises PMLA. In another embodiment, theLeu-Leu-Leu tripeptide is conjugated with the polymalic acid by an amidebond.

Various embodiments also include methods of decreasing the volume of aglioma in an individual, comprising providing a composition thatinhibits protein synthesis of Laminin-411, where the compositioncomprises PMLA covalently linked to a targeting antibody, a Laminin α4antisense polynucleotide and/or β1 antisense polynucleotide, and aLeu-Leu-Leu tripeptide, and administering the composition to theindividual at a concentration of up to but not exceeding 1 mg/mL. Inanother embodiment, the Laminin α4 antisense polynucleotide comprises a5′ to 3′ polynucleotide sequence characterized by SEQ. ID. NO.: 1. Inanother embodiment, the Laminin β1 antisense polynucleotide comprises a5′ to 3′ polynucleotide sequence characterized by SEQ. ID. NO.: 2. Inanother embodiment, the composition is administered systemically. Inanother embodiment, the composition is administered near the glioma. Inanother embodiment, the composition is administered by an intra-tumorinjection. In another embodiment, the composition is administered by animplantable device. In another embodiment, the composition isadministered subcutaneously, intraperitoneal and/or intravenously.

Other features and advantages of the invention will become apparent fromthe following detailed description, taken in conjunction with theaccompanying drawings, which illustrate, by way of example, variousembodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts an example of a polymalic acid based drug carrier. Thefigure depicts a schematic structure of a minimal Polycefin variant. Itcontains three components, the drug Morpholino oligonucleotide, theendosome escape unit, and a targeting monoclonal antibody. In addition,other drugs, such as siRNA, microRNA and aptamer, can be attached topolycefin via disulfide bond for cytoplasmic delivery.

FIG. 2 depicts the structure of poly(β-L-malic acid), or PMLA

FIG. 3 depicts the structure of PMLA-LLL. Structure of PMLA-LLL.

FIG. 4 depicts the structure of PMLA-(LeuOEt). Structure of PMLA-LeuOEt.

FIGS. 5 (a) and (b) depict charts of results for liposome leakage assayof different polymer conjugates with (a) describing results at a pH of7.4 and (b) describing results at a pH of 5.0. Concentration dependentliposome leakage by different PMLA conjugates at pH 7.4 and pH 5.0

FIGS. 6 (a) and (b) depicts charts demonstrating the effect of mPEG onliposome leakage with (a) describing results at a pH of 7.4 and (b)describing results at a pH of 5.0. Concentration dependent liposomeleakage by PMLA conjugates with and without mPEG at pH 7.4 and pH 5.0

FIG. 7 depicts a chart demonstrating the pH dependence differencebetween PMLA-LLL and PMLA-LeuOEt. pH dependent liposome leakage ofPMLA-LLL40% (50 μg/ml) (♦) and PMLA-LeuOEt40% (50 μg/ml) (▪).

FIG. 8 depicts a chart of pKa titration. Estimation of pKa values ofPMLA (▴), PMLA-LeuOEt (♦), and PMLA-LLL (▪) by the method of acid-basetitration. Only PMLA-LLL shows pKa of 5.5.

FIG. 9 depicts a chart of cell viability. Cell viability of glioma celllines U87MG and T98G after treatment with PMLA-LLL or PMLA-LeuOEt

FIG. 10 depicts western blot results, demonstrating cytoplasmic deliveryof antisense oligonucleotides. Synthesis of Laminin-411 alpha-4-chainsand beta-1-chains by human glioma cell lines U87MG and T98G aftertreatment with Polycefin variants.

FIG. 11 depicts the imaging of enhanced accumulation of PMLA-LLLcontaining transferrin receptor antibody at brain tumor Selectiveaccumulation in brain tumor of PMLA-LLL containing unspecific IgG(control) in panel A or anti-transferrin antibody (TfR mAb) in panel B.Fluorescence intensities are shown for the conjugated tracer in panel C.

FIG. 12 depicts a chart describing tumor volume after variousintravenous treatments (mm³, mean±SEM). Growth suppression of implantedhuman brain tumor after treatment with PBS (control) or differentPolycefin variants (Anova test, p<0.01).

DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Singleton et al., Dictionary of Microbiology and MolecularBiology 3^(rd) ed., J. Wiley & Sons (New York, N.Y. 2001); March,Advanced Organic Chemistry Reactions, Mechanisms and Structure 5^(th)ed., J. Wiley & Sons (New York, N.Y. 2001); and Sambrook and Russel,Molecular Cloning: A Laboratory Manual 3^(rd) ed., Cold Spring HarborLaboratory Press (Cold Spring Harbor, N.Y. 2001), provide one skilled inthe art with a general guide to many of the terms used in the presentapplication.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described.

As used herein, the term “laminin-411” also means “laminin-8.”

As used herein, the term “LLL” is an abbreviation ofL-Leu-(L-Leu)-(L-Leu).

As used herein, the term “PMLA” is an abbreviation for poly(β-L-malicacid).

As used herein, the term “PMLA-LLL” includes PMLA containing LLL, whichis conjugated by amide bond involving the N-terminal —NH₂.

As used herein, the term “PMLA-LLL40%” includes PMLA containing 40% ofpendant carboxylates (100%) conjugated by amide bond involving theN-terminal —NH₂ of oligopeptide trileucine LLL.

As used herein, the term “Polycefin” is a general name for therapeuticnanoconjugates based on polymalic acid for drug delivery. It may containmultifunctional components, such as a drug a targeting moiety, and anendosome escaping unit.

As used herein, the term “Polycefin-LLL” is a Polycefin variantcontaining 40% oligopeptide trileucine LLL conjugated as describedabove.

As used herein, the term “Polycefin-LeuOEt” is a Polycefin variantcontaining 40% leucine ethyl ester conjugated by amide bond to pendantcarboxylates (100%) involving the amino acid ester amino group.

As disclosed herein, naturally occurring PMLA, which is water-soluble,nontoxic, non-immunogenic and biodegradable, was investigated by theinventors for its potential for delivery of cytoplasmic antisenseoligonucleotides for cancer treatment. PMLA-LLL40% demonstratedpH-sensitive liposome leakage activity while PMLA containing 10% and 25%trileucine showed no or weak liposome leakage activity. A controlpolymer, PMLA containing 40% leucine ethyl ester (PMLA-LeuOEt40%) showedliposome leakage activity but in a pH independent manner. In addition,PMLA-LLL40% didn't show toxicity towards human glioma cell lines U87MGand T98G at concentration up to 1 mg/ml in cell viability studies, whilePMLA-LeuOEt40% was extremely toxic at high concentration. Therefore,PMLA-LLL40% was tested for cytoplasmic delivery of Morpholino antisensenucleotides against α4 and β1 chains of protein laminin-411, which isoverexpressed in gliomas and deposited in newly formed tumor bloodvessel basement membranes. Western blot analysis showed that PMLA-LLLcontaining α4 and β1 Morpholino antisense nucleotides remarkablyinhibited secretion of α4 and β1 protein chains of laminin-411 of U87MGand T98G cells, showing that this pH-sensitive Polycefin was able tosuccessfully deliver and liberate the antisense oligos into thecytoplasm.

As further disclosed herein, the Polycefin-LLL is designed to passthrough blood brain barrier (BBB) and contains three key components:antisense Morpholino oligonucleotides against laminin-411 α4 and β1chains, tandem targeting anti-transferrin receptor (TfR) antibodies, andnew pH-sensitive endosome escape unit, L-leucylleucylleucine(trileucine, LLL). Polycefin variants were injected intravenously ineach group of U-87MG cell inoculated mice (n=8 per group). Aftertreatment, the average tumor size of Polycefin-LLL and Polycefin-LeuOEtgroup was 4 mm³, and 18 mm³, respectively, compared with 47 mm³ in thegroup treated with PBS (p<0.01). The Polycefin-LLL nanoconjugate, whichis pH-sensitive, non-toxic, and biodegradable, proves to be the mosteffective for cytoplasmic delivery of active anticancer agents ascompared to previously described Polycefin variants (Lee, B.; BioconjugChem 2006, 17, (2), 317-26).

In one embodiment, the present invention provides a method of treating adisease in an individual by injecting intravenously Polycefin-LLL. Inone embodiment, the present invention provides a method of inhibitingexpression of a protein by administering a therapeutically effectiveamount of Polycefin-LLL. In another embodiment, the present inventionprovides a drug delivery molecule, comprising a polymerized carboxylicacid molecular scaffold covalently linked to L-leucylleucylleucine. Inanother embodiment, the Polycefin-LLL includes antisense morpholinooligos, targeting antibodies, and a pH-sensitive endosome escape unit.In another embodiment, the Polycefin-LLL contains 40% oligopeptidetrileucine. In another embodiment, the disease is cancer. In anotherembodiment, the antisense morpholino oligos include the antisense oflaminin-411 α4 and/or βI chains. In another embodiment, the targetingantibodies include tandem targeting anti-transferrin receptor (TfR)antibodies. In another embodiment, the pH sensitive endosome escape unitincludes L-leucylleucylleucine (trileucine, LLL). In another embodiment,the disease is treated by inhibiting expression of laminin-411. Inanother embodiment, the disease is treated by inhibition ofangiogenesis. In another embodiment, the individual is a human. Inanother embodiment, the individual is a mouse. In another embodiment,the Polycefin-LLL consists essentially of a polymerized carboxylic acidmolecular scaffold covalently linked to L-leucylleucylleucine, apH-sensitive endosome escape unit, a targeting antibody, and anantisense morpholino polynucleotide.

The present invention is also directed to a kit to prepare a drugdelivery molecule, as well as the delivery of a drug to the cytoplasm,and may include a polymerized carboxylic acid molecular scaffold, anantisense morpholino oligo, an siRNA, a micro-RNA, an aptamer, atargeting antibody, and L-leucylleucylleucine, and combinations thereof.The kit is an assemblage of materials or components, including least oneof the inventive compositions. Thus, in some embodiments the kitcontains a composition including PMLA-LLL, as described above.

The exact nature of the components configured in the inventive kitdepends on its intended purpose. For example, some embodiments areconfigured for the purpose of cytoplasmic delivery of active anticanceragents. In one embodiment, the kit is configured particularly for thepurpose of cytoplasmic drug delivery to mammalian subjects, such as, butnot limited to, human subjects, farm animals, domestic animals, andlaboratory animals.

Instructions for use may be included in the kit. “Instructions for use”typically include a tangible expression describing the technique to beemployed in using the components of the kit to effect a desired outcome,such as to prepare a PMLA-LLL nanoconjugate and to deliver a drug to acell cytoplasm. Optionally, the kit also contains other usefulcomponents, such as, diluents, buffers, pharmaceutically acceptablecarriers, syringes, catheters, applicators, pipetting or measuringtools, bandaging materials or other useful paraphernalia as will bereadily recognized by those of skill in the art.

The materials or components assembled in the kit can be provided to thepractitioner stored in any convenient and suitable ways that preservetheir operability and utility. For example the components can be indissolved, dehydrated, or lyophilized form; they can be provided atroom, refrigerated or frozen temperatures. The components are typicallycontained in suitable packaging material(s). As employed herein, thephrase “packaging material” refers to one or more physical structuresused to house the contents of the kit, such as inventive compositionsand the like. The packaging material is constructed by well knownmethods, preferably to provide a sterile, contaminant-free environment.The packaging materials employed in the kit are those customarilyutilized in preparing a nanoconjugate. As used herein, the term“package” refers to a suitable solid matrix or material such as glass,plastic, paper, foil, and the like, capable of holding the individualkit components. Thus, for example, a package can be a glass vial used tocontain suitable quantities of an inventive composition containing asolution of PMLA-LLL or components thereof. The packaging materialgenerally has an external label which indicates the contents and/orpurpose of the kit and/or its components.

In various embodiments, the present invention provides pharmaceuticalcompositions including a pharmaceutically acceptable excipient alongwith a therapeutically effective amount of Polycefin-LLL.“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients may be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

In various embodiments, the pharmaceutical compositions according to theinvention may be formulated for delivery via any route ofadministration. “Route of administration” may refer to anyadministration pathway known in the art, including but not limited to anintravenous injection, aerosol, nasal, oral, transmucosal, transdermalor parenteral. “Parenteral” refers to a route of administration that isgenerally associated with injection, including intraorbital, infusion,intraarterial, intracapsular, intracardiac, intradermal, intramuscular,intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal,intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous,transmucosal, or transtracheal. Via the parenteral route, thecompositions may be in the form of solutions or suspensions for infusionor for injection, or as lyophilized powders.

The pharmaceutical compositions according to the invention can alsocontain any pharmaceutically acceptable carrier. “Pharmaceuticallyacceptable carrier” as used herein refers to a pharmaceuticallyacceptable material, composition, or vehicle that is involved incarrying or transporting a compound of interest from one tissue, organ,or portion of the body to another tissue, organ, or portion of the body.For example, the carrier may be a liquid or solid filler, diluent,excipient, solvent, or encapsulating material, or a combination thereof.Each component of the carrier must be “pharmaceutically acceptable” inthat it must be compatible with the other ingredients of theformulation. It must also be suitable for use in contact with anytissues or organs with which it may come in contact, meaning that itmust not carry a risk of toxicity, irritation, allergic response,immunogenicity, or any other complication that excessively outweighs itstherapeutic benefits.

The pharmaceutical compositions according to the invention can also beencapsulated, tableted or prepared in an emulsion or syrup for oraladministration. Pharmaceutically acceptable solid or liquid carriers maybe added to enhance or stabilize the composition, or to facilitatepreparation of the composition. Liquid carriers include syrup, peanutoil, olive oil, glycerin, saline, alcohols and water. Solid carriersinclude starch, lactose, calcium sulfate, dihydrate, terra alba,magnesium stearate or stearic acid, talc, pectin, acacia, agar orgelatin. The carrier may also include a sustained release material suchas glyceryl monostearate or glyceryl distearate, alone or with a wax.

The pharmaceutical preparations are made following the conventionaltechniques of pharmacy involving milling, mixing, granulation, andcompressing, when necessary, for tablet forms; or milling, mixing andfilling for hard gelatin capsule forms. When a liquid carrier is used,the preparation will be in the form of a syrup, elixir, emulsion or anaqueous or non-aqueous suspension. Such a liquid formulation may beadministered directly p.o. or filled into a soft gelatin capsule.

The pharmaceutical compositions according to the invention may bedelivered in a therapeutically effective amount. The precisetherapeutically effective amount is that amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given subject. This amount will vary depending upon a variety offactors, including but not limited to the characteristics of thetherapeutic compound (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type and stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication), the nature of the pharmaceutically acceptable carrier orcarriers in the formulation, and the route of administration. Oneskilled in the clinical and pharmacological arts will be able todetermine a therapeutically effective amount through routineexperimentation, for instance, by monitoring a subject's response toadministration of a compound and adjusting the dosage accordingly. Foradditional guidance, see Remington: The Science and Practice of Pharmacy(Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

Typical dosages of an effective cytoplasmic drug delivery can be in theranges recommended by the manufacturer where known therapeutic compoundsare used, and also as indicated to the skilled artisan by the in vitroresponses or responses in animal models. Such dosages typically can bereduced by up to about one order of magnitude in concentration or amountwithout losing the relevant biological activity. Thus, the actual dosagewill depend upon the judgment of the physician, the condition of thepatient, and the effectiveness of the therapeutic method based, forexample, on the in vitro responsiveness of the relevant primary culturedcells or histocultured tissue sample, such as biopsied malignant tumors,or the responses observed in the appropriate animal models, aspreviously described.

As described herein, various embodiments of the invention include thedelivery of a drug to the cytoplasm. As readily apparent to one of skillin the art, the invention may be applied to any number of targets whereit would be beneficial to deliver a drug or molecule to the cytoplasmand the invention is in no way limited to nucleotides coding forlaminin-411 α4 and/or β1 chains. Similarly, any number of conditionsand/or diseases may be beneficially treated and the invention is in noway limited to treatment of cancer, tumor suppression and/or inhibitionof glioblastoma angiogenesis. For example, various embodiments describedherein may include the treatment of HIV and/or AIDS, and any othernumber of conditions where it is advantageous to deliver a drug to thecytoplasm. Finally, as would be readily apparent to one of skill in theart, various molecules and/or drugs may also be delivered to thecytoplasm, including the delivery of proteins, and the variousembodiments described herein are in no way limited to delivery ofantisense morpholino oligo and/or other oligonucleotides. As known toone of skill in the art, protein therapy is often difficult as theprotein is unstable or degraded in the liposome before it can beeffective, whereas various embodiments described herein overcome thesedifficulties by allowing deliver of the protein to the cytoplasm.

Various embodiments of the invention may also be practiced inconjunction with an overall treatment regimen. For example, as describedherein, various embodiments include the delivery of a drug to thecytoplasm by way of disruption of the endosome. As readily apparent toone of skill in the art, additional drugs or substances that werepreviously inactive in the endosome will then become active upon thedisruption of the endosome. Thus, various embodiments of the inventionmay include additional drugs or substances administered to the subjectbeing treated and the invention is not only limited to drugs and/ormolecules covalently linked to the scaffold as described herein.Similarly, as readily apparent to one of skill in the art, variousembodiments of the invention may be used in conjunction with or incombination with additional therapeutics.

As also described herein, various embodiments include intravenousinjection of Polycefin-LLL. Intravenous injection provides numerousadvantages over most current procedures required for drug delivery pastthe blood brain barrier. For example, intravenous injection is a lessinvasive procedure than intracranial injection. However, the inventionis in no way limited to intravenous injection and may be administered byany number of methods. For example, the composition may be administereddirectly in an intra-tumor injection, or by an implantable device,subcutaneously, intraperitoneal, intravenously, or any other methods ofadministration readily apparent to one of skill in the art.

As readily apparent to one of skill in the art, various embodimentsdescribed herein may also be used in conjunction with delivery of siRNA,micro-RNA, and aptamers. PMLA-LLL may act as a transfection agent forcytoplasmic delivery of siRNA, micro-RNA, and aptamers.

Numerous varieties of PMLA-LLL may also be effectively used. Forexample, liposome leakage activity may be effected by pH levels, ormodified due to a higher content of LLL, or other tripeptides, orcombination of two or more tripeptides, as well as the length of peptideside chain. Thus, as readily apparent to one of skill in the art, thecytoplasmic drug delivery molecule described in various embodimentsherein may be modified depending upon the desired purpose of use andassociated conditions, and the invention is not limited to onlyPMLA-LLL40%.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described. For purposes ofthe present invention, the following terms are defined below.

EXAMPLES

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention.

Example 1 General

Naturally occurring poly(β-L-malic acid) (PMLA) is water-soluble,nontoxic, non-immunogenic and biodegradable. As described herein, theinventors investigated its potential for use of cytoplasmic delivery ofantisense oligonucleotides for cancer treatment. PMLA containing 40%oligopeptide trileucine (PMLA-LLL40%) demonstrated pH-sensitive liposomeleakage activity while PMLA containing 10% and 25% trileucine showednone or weak liposome leakage activity. A control polymer, PMLAcontaining 40% leucine ethyl ester (PMLA-LeuOEt40%) also showed Liposomeleakage activity but in a pH independent manner. In addition,PMLA-LLL40% didn't show toxicity towards human glioma U87MG and T98Gcells at concentration up to 1 mg/ml in cell viability studies, whilePMLA-LeuOEt40% was extremely toxic at high concentration. Therefore,PMLA-LLL40% was tested for cytoplasmic delivery of Morpholino antisensenucleotides against α4 and β1 chains of protein laminin-411, which isoverexpressed in gliomas and deposited in newly formed tumor bloodvessel basement membranes. By western blot analysis Polycefin-LLLcontaining α4 and β1 Morpholino antisense nucleotides markedly inhibitedthe expression of α4 and β1 protein chains of laminin-411 of U87MGcells, showing that this pH-sensitive Polycefin was able to successfullydeliver and liberate the antisense oligos into the cytoplasm. Thisversion of Polycefin (Polycefin-LLL) designed to pass through the bloodbrain barrier (BBB) contains three key components: antisense Morpholinooligos against laminin-411 α4 and β1 chains, tandem targetinganti-transferrin receptor (TfR) antibodies, and the new pH-sensitiveendosome escape unit, L-leucylleucylleucine (trileucine, LLL). Polycefinvariants were injected intravenously in each group of U87MG cellinoculated mice (n=8 per group). After treatment, the average tumor sizeof Polycefin-LLL40% group was 4 mm³, compared to 18 mm³ in the grouptreated with Polycefin-LeuOEt40% (the original version of Polycefin),and 47 mm³ in the group treated with PBS (p<0.01). The newly designedPolycefin-LLL nanoconjugate, which is pH-sensitive, non-toxic, andbiodegradable, proved the most effective for cytoplasmic delivery ofactive anticancer agents.

Example 2 Materials

Morpholino-3′ —NH₂ antisense oligonucleotides SEQ. ID. NO.: 1 to laminin(MORPH-AON-1) and SEQ. ID. NO.: 2 to laminin β1 (MORPH-AON-2) chainswere custom-made by Gene Tools (USA). Rat antimouse monoclonal TfRantibody R17217 (mTfR) and mouse antihuman monoclonal TfR antibodyRVS-10 (huTfR) were purchased from Southern Biotech (USA).Poly(β-L-malie acid) (PMLA) (Mw, 100,00; polydispersity 1.3) wasobtained from culture broth of Physarum polycephalum and was purifiedand size-fractionated by chromatography. mPEG5000-amine andmaleimide-PEG3400-maleimide were obtained from Laysan Bio, Inc. (USA).H-Leu-Leu-OH and H-Leu-Leu-Leu-OH were purchased from Bachem Americas,Inc. (USA). Glioma cell lines U87MG and T98G were obtained from theAmerican Type Culture Collection (USA).

Example 3 Synthesis of PMLA Trileucine Conjugates PMLA-LLL40% andPMLA-LLL40%-MEA

To a 1 ml solution of PIMA 73 mg (0.63 mmol equivalent to malic acid) inacetone was added a mixture of N-hydroxysuccinimide (NHS) anddicyclohexylcarbodiimide (DCC) in 2 ml DMF. After 4 hr stirring at roomtemperature, dicyclohexylurea was removed by filtration and the volumeof reaction solution was reduced to about 0.5 ml with evaporation.Subsequently, to the reaction mixture was added 2 ml pyridine and asolution of H-Leu-Leu-Leu-OH (90 mg, 0.25 mmol, 40% equivalent to thetotal malyl groups), dissolved with the assistance of 24 μltrifluoacetic acid) in DMF. After 2 hr stirring at room temperature,triethylamine 20 μl was added to the reaction mixture. The completion ofconjugation was verified with TLC with ninhydrin test. The unreactedN-hydroxysuccinimidyl ester was hydrolysed by the addition of water. Thefinal product PMLA-LLL40% was purified with PD-10 column (GEHealthcare).

To prepare PMLA-LLL40%-MEA, after the reaction of LLL with PMLA wascomplete, the unreacted N-hydroxysuccinimidyl ester was not hydrolysed.Instead, 2-mercaptoethylamine hydrochloride (7.2 mg, 0.06 mmol, 10%equivalent to the total malyl groups) (MEA) and triethylamine 9 μl wasadded to the reaction mixture. The reaction finished within 30 min andthe completion of conjugation was verified with TLC with ninhydrin test.Then, the unreacted N-hydroxysuccinimidyl ester was hydrolysed by theaddition of water. The final product PMLA-LLL40%-MEA was purified withPD-10 column (GE Healthcare).

Example 4 Synthesis of Polycefin-LLL and Polycefin-LeuOEt

The conjugation of Morpholino oligonuclecotides, antibodies andfluorescence dye to PMLA-LLL40%-MEA and PMLA-LeuOEt40%-MEA to obtainPolycefin-LeuOEt and Polycefin-LLL was described previously (Lee, B.;Bioconjug Chem 2006, 17, (2), 317-26).

Example 5 Lipsome Leakage Assay

Liposome was prepared with extrusion method. Briefly, the mixture of eggPC and cholesterol (molar ratio, 2:1) dissolved in CHCl₃/MeOH (v/v, 2:1)was dried under a stream of nitrogen. The lipid mixture was hydratedwith HBS buffer (5 mM HEPES, 150 mM NaCl, pH 7.4) containing 90 mMcalcein, followed by 19 extrusions through 0.1 μm polycarbonate membraneusing mini-extruder (Avanti Polar Lipids). Samples with serial dilutionswere dissolved in two 95 μl buffers of different pH in a plate, 137 mMHEPES buffer pH 7.4 and 137 mM citrate buffer pH 5.0, Liposome 5 μl(lipid concentration 160 μM) was added to each sample and the plate wasincubated at room temperature for 1 hr. Complete leakage of calcein wasachieved with the addition of 0.25% (v/v) triton-X 100 solution ofrespective buffers. The calcein release was measured with fluorometerwith excitation wavelength 488 nm and emission wavelength 535 nm.

Example 6 Cytotoxicity Studies

U87MG and T98G cells were seeded in a 96-well plate (10,000 cells/wellin 100 μL media) and incubated for 24 h. Then the cells were incubatedwith polymers of different concentrations in 200 μL of media for another24 h. The viable cells were quantified using the CellTiter 96 AqueousOne Solution Cell Proliferation Assay kit (Promega) by reading theabsorbance at 490 nm with a Spectra Max Plus 384 ELBA reader.

Example 7 Acid-Base Titration

The pKa values of PMLA, PMLA-LLL40%, and PMLA-LeuOEt40% were estimatedwith acid-base titration. Polymer conjugates 16 mg were dissolved in 8ml of deionized water and the pH of solutions were adjusted to low value(around 2). The polymer solutions were titrated with 0.5 N NaOH in 5 μlaliquots. The pH value of polymer solutions after each addition of NaOHwas measured after careful mixing and sufficient equilibration.

Example 8 Confocal Microscopy

U87MG cells were plated in a 8 well Lab-Tek II chamber slide containing200 μl Eagle's MEM with 10% fetal calf serum, Glutamine, sodiumbicarbonate, nonessential amino acids, antibiotics, and sodium pyruvate.The cells were incubated with Polycefin-LLL-(SS-Rh) or Polycefin-LLL-Rhwith RH (=rhodamine, SS-RH=disulfide conjugated RH) concentration 2 μMfor 24 h, 6 h, 2 h, and 0 h. The cells were washed with Dulbecco'smodified phosphate-buffered saline (DPBS) for 5 times and fixed with 2%paraformaldehyde for 10 min followed by washing with DNS three times.The cells were visualized with TCS SP spectral scanner (LeicaMicrosystems, Mannheim, Germany).

Example 9 Western Blotting

Human glioma U87MG and T98 produce secreted laminin 411. Polycefin-LLLor the other variants at a concentration of 1.4 μM MORPH-AON was addedto the culture medium of these cultured cell lines on day 1 and 4. Forthe detection of laminins, serum-free conditioned medium was sampled atday 6 from culture supernatants above equal numbers of cells that hasbeen cultured for the same period of time. Samples were concentrated10-fold by filtering through Centriplus filtration devices (Millipore,Bedford, Mass.) and proteins were separated using Tris-Glycine 8%SDS-PAGE (Invitrogen) under reducing conditions. The gels were blottedonto nitrocellulose membrane (Invitrogen). The membranes were probedwith mAbs followed by chemiluminescent detection using the Immune-Starkit with alkaline phosphatase-conjugated secondary antibodies (Bio-RadHercules, Calif.). Antibodies were used to laminin α4 [mAb 8B12] and βIchains (mAb LT3). Antibody to human fibronectin 8^(th) type III repeat[mAb 568] was used to control for equal loading of gel lanes.

Example 10 Imaging

The imaging procedures for drug accumulation in mice tumors were same aspreviously reported.³⁰ Briefly, 5×10⁴ of U87MG human glioblastoma cellswere stereotactically implanted into the right basal ganglia field ofathymic mice (CrTac:NCr-Foxnlnu Homozygous, Taconic, USA). At day 2Iafter tumor implantation, 100 μl of AlexaFluor 680 labeled Polycefinvariants was injected intravenously at the concentration of 3 μM. Forassessment of drug distribution and localization in nude mice, XenogenIVIS 200 was used under the Isoflurane anesthesia at different timepoints (before drug administration; 1 h, 3 h, 6 h, 24 h, 48 h after drugadministration). Twenty-four hours after drug administration, the micewere euthanized and the circulating drugs in blood vessels wereeliminated by intraarterial PBS perfusion for 20 min. The brain washarvested to detect the fluorescent signal. The fluorescent signalintensities in the tumor, tumor adjacent area, normal cerebrum andnormal cerebellum were analyzed by Xenogen Living Image, Version 2.50(WaveMetrix, USA). All animal procedures were carried out in accordancewith IACUC protocols approved by animal welfare committee atCedars-Sinai Medical Center.

Example 11 Tumor Suppression

U87MG human glioblastoma cells (5×10⁴) were stereotactically implantedinto the right basal ganglia field of athymic mice (n=8 per group)(CrTac:NCr-Foxnlnu Homozygous, Taconic, USA). After day 8, Polycefinvariants were injected intravenously at dose of 5 mg/kg AONs tolaminin-411 α4 and β1 chains every three days for 8 injections in total.Mice in all Polycefin- and PBS-treated groups were sacrificed on day 48after tumor cell inoculation and tumor size was measured.

Example 12 Results—Synthesis of PMLA-LLL

The synthesis of PMLA-LLL is simple conjugation of polymalic withtrileucine peptide. The carboxyl group of PMLA was activated with DCCand NHS to form N-hydroxysuccinimidyl esters which is substituted by theamino group of trileucine. Trileucine, however, is not readily dissolvedin reaction compatible solvent such as DMF and DMSO. To solve this, itwas first dissolved in DMF with the assistance of 1.25 equivalent oftrifluoroacetic acid and excess of pyridine was used as base. Thecompleteness of the reaction was monitored with ninhydrin test. Theaddition of triethyl amine was to facilitate the completion of thereaction.

Example 13 Results—Liposome Leakage Assay

Liposome leakage assay was used for the membrane destabilizing activityof various PMLA conjugates. PMLA conjugates containing 10%, 25% and 40%of trileucine were prepared and their membrane destabilizing activitywas evaluated using liposome leakage assay. PMLA-LLL10% didn't showliposome leakage at the tested concentrations at both pH 7.4 and pH 5.0.PMLA-LLL25% and PMLA-LLL40% showed little liposome leakage activity evenat concentration as high as 2 mg/ml (less than 10% leakage) at pH 7.4.In contrast, PMLA-LLL40% showed significant liposome leakage at pH 5.0from low concentration (1 μg/ml) to high concentration (2 mg/ml) andabout 50% liposome leakage was observed at concentration of 20 μg/ml.PMLA-LLL25%, however, only showed Liposome leakage activity at highconcentration. Therefore, the content of LLL is important in modulatingthe liposome activity of PMLA-LLL conjugates: the higher the content ofLLL, the more efficient for liposome leakage. To see the length effectof peptide chain on liposome leakage activity, PMLA conjugatescontaining dileucine LL40% and tetraleucine LLLL40% were also tested.However, neither conjugates (PMLA-LL40%) and (PMLA-LLLL40%) showedsignificant liposome leakage at both pH 7.4 and pH 5.0, showing that thelength of peptide chain is also important for the liposome leakageactivity, with the chain length of the tripeptide optimal for liposomeleakage.

Meanwhile, liposome leakage was also evaluated for a control polymerPMLA-LeuOEt40%, a PMLA conjugate containing 40% leucine ethyl esterpreviously proposed for endosome disruption. At pH 5.0, PMLA-LeuOEt40%showed a similar liposome leakage profile to that of PMLA-LLL40%, butwith less liposome activity at low concentration and with higheractivity at high concentration compared with PMLA-LLL40%. UnlikePMLA-PMLA-LeuOEt40% also showed good liposome leakage activity at pH7.4. Consequently, the liposome leakage activity of PMLA-LLL40% is pHsensitive, while that of PMLA-LeuOEt40% is pH insensitive.

Therefore, the pH sensitivity of liposome leakage was also evaluated forboth PMLA-LLL40% and PMLA-LeuOEt40%. Liposome leakage activity of eachpolymer was tested at concentration of 50 μg/ml and at pH values rangingfrom 5.0 to 7.5, representing the pH values which polymers willencounter during endocytosis. PMLA-LLL40% virtually showed no liposomeleakage activity at pH 7 and 7.5 and started to show slight activity atpH 6 and 6.5. When pH is lower than 5.5, strong liposome leakage wasobserved (>60%). PMLA-LeuOEt40% demonstrated strong liposome leakageactivity (>80%) at all pHs tested, showing it can destabilize membranenot only at endosomal pH but also at physiological pH. Therefore, theliposome leakage activity of PMLA-LLL is pH sensitive; while that ofPMLA-LeuOEt is pH insensitive. The pH insensitivity of PMLA-LeuOEt maycause the cytotoxocity due to its capability to destabilize cellmembrane at physiological pH.

Example 14 Results—Effect of mPEG on Liposome Leakage

Polyethyleneglycol (PEG) is widely used for protection of degradation ofnanoconjugates, and previously it was also used for the protection ofPMLA conjugate. However, how the PEGylation or PMLA may affect themembrane destabilization activity of the whole conjugate is unknown. Itis necessary to resolve this to optimize the cytoplasmic deliverycapability of newly designed conjugate. PEGylation had virtually noeffect on the liposome leakage of PMLA-LLL40% at pH 7.4; however, at pH5.0, more than 10-fold of PMLA-LLL-mPEG has to be used to achieve sameamount of liposome leakage induced by PMLA-LLL. In contrast, theliposome activity of PMLA-LeuOEt was affected by PEGylation at both pH7.4 and pH 5.0. In addition, the PEGylation of PMLA-LeuOEt had a morenegative effect on liposome leakage. At both pHs, more than 100-fold ofPMLA-LeuOEt-mPEG has to be used to achieve same amount of Liposomeleakage induced by PMLA-LeuOEt.

Example 15 Results—Cytotoxicity

To evaluate the cytotoxicity of PMLA-LLL40% and PMLA-LeuOEt40%, twohuman glioma cell lines, U87MG and T98G, were treated with eachconjugate of different concentrations. Viable cells were determinedusing CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit(Promega). PMLA-LLL/40% didn't show any cytotoxicity towards both celllines at all concentrations tested up to 1 mg/ml. AlthoughPMAL-LeuOEt40% didn't show significant cytoxicity at concentrationslower than 100 μg/ml, its cytotoxicity increased drastically atconcentrations higher than 125 μg/ml for U87MG cells and higher than 500μg/ml for T980 cells. After the treatment of cells with polymers (500μg/ml) for 24 h, U87MG cells treated with PMLA-LLL40% looked healthywithout any morphological changes. Instead, cells treated withPMLA-LeuOEt40% at toxic concentration were all dead and showed cellshapes typical of necrosis. Based on cytotoxicity results, PMLA-LLL40%is safer than PMLA-LeuOEt40% when used in drug delivery.

Example 16 Results—Detection of Laminin Expression by Western BlotAnalysts

Western blot was used to confirm that the newly designed nanoconjugatecan achieve cytoplasmic delivery of antisense oligonucleotides. Culturesof two human glioma cell lines, U87MG and T98G were treated withPolycefin variants. Polycefin-LeuOEt and Polycefin-LLL are PMLAconjugates containing the nonspecific membrane escape unit LeuOEt or theendosome specific escape unit LLL and, in addition, Morpholino antisenseoligos, anti-transferrin receptor antibodies. Inhibition of laminin-411synthesis by Polycefins was determined by western blotting. Fibronectinwas used as an internal reference to correct for equal loading ofsamples. In the absence of endosome escape unit, PMLA conjugated withMorpholino oligo only (lane 2) or with PMLA-(TfR mAb)-AON (lane 3), didnnot significantly inhibit the synthesis α4 and β1 chains of lamini-411compared with no treatment (lane 1). Both Polycefin-LeuOEt (lane 4) andPolycefin-LLL (lane 5) inhibited the synthesis of laminin-411.Polycefin-LLL showed the strongest inhibition. The inhibition byPolycefin-LLL is in accordance with cytoplasmic delivery of Morpholinooligos. The results demonstrate the safe and efficient delivery ofnucleic acid based drugs by Polycefin-LLL.

Example 17 Results—Selective Accumulation of Polycefin in Tumor

To confirm that Polycefin-LLL crossed selectively blood-brain-tumorbarrier with the result to be selectively accumulated only in braintumor, the inventors carried out intravenous injections into human braintumor-bearing nude mice and monitored its distribution. In theseexperiments, the inventors used Alexa Fluor 680 conjugated PMLA-LLL40%containing transferrin receptor antibody. In control experiments,transferrin receptor antibody was replaced by unspecific mouse IgG.Xenogen IVIS 200 imaging system was used to detect the drug distributionin whole body and isolated mouse organs 24 h after injection. Repeatedexperiments were conducted, giving qualitatively the same results. Asshown in FIG. 11, PMLA-LLL40% containing transferrin receptor antibodywas selectively accumulated at the tumor site, and its accumulation wassignificantly higher than that of the control conjugate containing thenonspecific IgG after 24 (P<0.01).

Example 18 Results—Tumor Suppression Study

Laminin-411 is associated with glioma recurrence and shorter patientsurvival time, and it is important for glioblastoma angiogenesis. Invivo inhibition of laminin-411, therefore, provides a safe and effectiveway to suppress tumor growth. Mice were treated with two Polycefinvariants, Polycefin-LeuOEt and Polycefin-LLL and were sacrificed on day48 after tumor cell inoculation. After sacrifice of mice, their tumorsize was measured. Treatment with Polycefin-LeuOEt significantlysuppressed the brain tumor growth with average tumor size of 18 mm³compared with 47 mm³ in the control group treated with PBS (one wayAnova test, p<0.01). Moreover, the treatment with Polycefin-LLLsuppressed the tumor growth to an even larger extent at highsignificance (average tumor size 4 mm³, p<0.001). The results show thatPolycefin-LLL delivers anticancer agents not only in vitro but also invivo. Because this carrier favors delivery through endosomes it isconsidered a safe and efficient cytoplasmic device for delivery ofnucleic acid based drugs and proteins.

Various embodiments of the invention are described above in theDescription of the Invention. While these descriptions directly describethe above embodiments, it is understood that those skilled in the artmay conceive modifications and/or variations to the specific embodimentsshown and described herein. Any such modifications or variations thatfall within the purview of this description are intended to be includedtherein as well. Unless specifically noted, it is the intention of theinventor that the words and phrases in the specification and claims begiven the ordinary and accustomed meanings to those of ordinary skill inthe applicable art(s).

The foregoing description of various embodiments of the invention knownto the applicant at this time of filing the application has beenpresented and is intended for the purposes of illustration anddescription. The present description is not intended to be exhaustivenor limit the invention to the precise form disclosed and manymodifications and variations are possible in the light of the aboveteachings. The embodiments described serve to explain the principles ofthe invention and its practical application and to enable others skilledin the art to utilize the invention in various embodiments and withvarious modifications as are suited to the particular use contemplated.Therefore, it is intended that the invention not be limited to theparticular embodiments disclosed for carrying out the invention.

While particular embodiments of the present invention have been shownand described, it will be obvious to those skilled in the art that,based upon the teachings herein, changes and modifications may be madewithout departing from this invention and its broader aspects and,therefore, the appended claims are to encompass within their scope allsuch changes and modifications as are within the true spirit and scopeof this invention. Furthermore, it is to be understood that theinvention is solely defined by the appended claims. It will beunderstood by those within the art that, in general, terms used herein,and especially in the appended claims (e.g., bodies of the appendedclaims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to inventions containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should typically be interpreted to mean “atleast one” or “one or more”); the same holds true for the use ofdefinite articles used to introduce claim recitations. In addition, evenif a specific number of an introduced claim recitation is explicitlyrecited, those skilled in the art will recognize that such recitationshould typically be interpreted to mean at least the recited number(e.g., the bare recitation of “two recitations,” without othermodifiers, typically means at least two recitations, or two or morerecitations).

Accordingly, the invention is not limited except as by the appendedclaims.

1. A drug delivery system, comprising: a polymerized polymalic acidmolecular scaffold having a plurality of pendant carboxyl groups and oneor more biologically active molecular modules, each of the one or morebiologically active molecular module is covalently linked to a pendantcarboxyl group of the polymerized polymalic acid molecular scaffold,wherein one of the one or more biologically active molecular modulescomprises an endosome escape unit, the endosome escape unit comprisesL-leucylleucylleucine (LLL).
 2. The drug delivery system of claim 1,wherein the endosome escape unit is configured to cause disruption of60%, or more of endosome membranes at a pH of 5.5, or lower.
 3. The drugdelivery system of claim 1, wherein one of the one or more biologicallyactive molecular modules comprises a siRNA, a microRNA, and/or anaptamer.
 4. The drug delivery system of claim 1, whereinL-leucylleucylleucine (LLL) is conjugated to 10% or more of the pendantcarboxyl groups of the polymerized polymalic acid molecular scaffold. 5.The drug delivery system of claim 1, wherein L-leucylleucylleucine (LLL)is conjugated to 40% or more of the pendant carboxyl groups of thepolymerized polymalic acid molecular scaffold.